The Zn3 domain of human poly(ADP-ribose) polymerase-1 (PARP-1) functions in both DNA-dependent poly(ADP-ribose) synthesis activity and chromatin compaction

Abstract

PARP-1 is involved in multiple cellular processes, including transcription, DNA repair, and apoptosis. PARP-1 attaches ADP-ribose units to target proteins, including itself as a post-translational modification that can change the biochemical properties of target proteins and mediate recruitment of proteins to sites of poly(ADP-ribose) synthesis. Independent of its catalytic activity, PARP-1 binds to chromatin and promotes compaction affecting RNA polymerase II transcription. PARP-1 has a modular structure composed of six independent domains. Two homologous zinc fingers, Zn1 and Zn2, form the DNA-binding module. Zn1-Zn2 binding to DNA breaks triggers catalytic activity. Recently, we have identified a third zinc binding domain in PARP-1, the Zn3 domain, which is essential for DNA-dependent PARP-1 activity. The crystal structure of the Zn3 domain revealed a novel zinc-ribbon fold and a homodimeric Zn3 structure that formed in the crystal lattice. Structure-guided mutagenesis was used here to investigate the roles of these two features of the Zn3 domain. Our results indicate that the zinc-ribbon fold of the Zn3 domain mediates an interdomain contact crucial to assembly of the DNA-activated conformation of PARP-1. In contrast, residues located at the Zn3 dimer interface are not required for DNA-dependent activation but rather make important contributions to the chromatin compaction activity of PARP-1. Thus, the Zn3 domain has dual roles in regulating the functions of PARP-1.

FIGURE 1.

PARP-1 has a modular structure composed of six domains. A, schematic representation of PARP-1 domain structure. B, ribbon representation of the x-ray structure of the Zn3 domain (30). C, crystallographic dimer of the Zn3 domain. One Zn3 monomer is drawn in green, and the second monomer is drawn in orange. The transparent dotted line highlights the Zn3 dimer interface.

FIGURE 2.

The zinc-ribbon fold of the Zn3 domain is essential for PARP-1 DNA-dependent activity. A, x-ray structure of the zinc-ribbon fold of the PARP-1 Zn3 domain. Residues mutated in B–D are drawn as sticks. Mutation of residues colored red led to a defect in DNA-dependent activity, although mutation of residues colored green had no affect on DNA-dependent activity. B and C, DNA-dependent automodification activity of WT PARP-1 or mutants (0.62 μm) with 1 μm duplex DNA and 5 mm NAD⁺. The indicated time points were analyzed by 12% SDS-PAGE. D, radioactive automodification assay. Top, WT PARP-1 and mutants (0.5 μm) were incubated with 1 μm duplex DNA and radiolabeled NAD⁺ (0.2 μm). Phosphorimage analysis of a 12% SDS-PAGE is shown. Bottom, same experiment was performed in the presence of unlabeled NAD⁺ as a control for the amount of the enzymes used in the reaction. The SDS-PAGE was treated with Imperial protein stain.

FIGURE 3.

The zinc-ribbon fold of the Zn3 domain of PARP-1 is necessary for NAD⁺-dependent transcriptional derepression. Results of ERα-dependent in vitro transcription assays with chromatin templates are shown. The pERE plasmid DNA template was assembled into chromatin using ACF and subjected to in vitro transcription under the conditions shown. A, representative gel image of the in vitro transcription assay with WT, W318R, and E988A PARP-1 proteins in the presence or absence of NAD⁺. E2, 17β-estradiol, B, quantification of three independent in vitro transcription assays. Each bar represents the mean ± S.E.

FIGURE 4.

Zn3 domain added in trans restores activity of a PARP-1 ΔZn3 deletion mutant. A, DNA-dependent automodification activity of PARP-1 ΔZn3 (0.62 μm) is restored by the addition of the WT Zn3 domain (0.62 μm, but not by the W318R mutant of the Zn3 domain (0.62 μm). 1 μm duplex DNA and 5 mm NAD⁺ were used. The indicated time points were analyzed by SDS-PAGE (15%) stained with Imperial protein stain. B, DNA-dependent automodification activity of WT PARP-1 (0.62 μm), E988A, and W318R mutants (0.62 μm in individual experiments, 0.31 μm each in combination experiment) on 7.5% SDS-PAGE.

FIGURE 5.

Mutations at the Zn3 dimer interface do not disrupt PARP-1 DNA-dependent automodification activity. A, ribbon representation of the Zn3 dimer with mutated residues drawn as blue sticks and labeled on each monomer. The N terminus is labeled n, and the C terminus is residue Pro-359. B, DNA-dependent automodification activity of WT PARP-1 and Zn3 dimer mutants (0.62 μm) with 1 μm duplex DNA and 5 mm NAD⁺. See supplemental Fig. S5 for additional Zn3 dimer interface mutants.

FIGURE 6.

Zn3 dimer interface mutations are deficient in the ability to compact chromatin. MNase protection assay with chromatin templates. The pERE plasmid DNA template was assembled into chromatin using ACF and subjected to MNase digestion under the conditions shown. A, representative gel image of the MNase protection assay with WT PARP-1 and five mutant PARP-1 proteins. Shown is a 2-fold serial dilution for each of the PARP-1 proteins at a final concentration of 33, 66, and 132 nm. The control lane indicates the amount of digestion in the absence of PARP-1 protein. The amount of MNase used in the assay was empirically determined. M, 123-bp molecular weight ladder. B, quantification of three MNase protection assays. Arrowheads in A indicate the MNase-resistant bands that were quantified. Percentages determined are relative to WT PARP-1 at the highest concentration used. Each bar represents the mean ± S.E.

FIGURE 7.

Zn3 mutations in the dimer interface region are unable to fully repress transcription. Results of ERα-dependent in vitro transcription assays with chromatin templates are shown. The pERE plasmid DNA template was assembled into chromatin using ACF and subjected to in vitro transcription under the conditions shown. A, in vitro transcription assay with WT PARP-1 and four Zn3 dimer interface mutants (Q241L, LDVD, PGPG, and F357Y) and one zinc-ribbon fold mutant (W318R). ERα and 17β-estradiol (E2) were used to activate transcription. Quantification of the relative levels of transcription is indicated below the image of the representative gel. B, quantification of three independent in vitro transcription assays. Each bar represents the mean ± S.E.

FIGURE 8.

X-ray and NMR structures of the Zn3 domain demonstrate different positions of the C-terminal tail. A, comparison of the x-ray and NMR models of the Zn3 domain. Structures were superimposed by aligning the first three α-helices of the Zn3 domain. The relative orientation of the zinc-ribbon fold is rotated ∼60° (bottom arrow), and the C terminus of the Zn3 domain is in two different conformations (top arrow). B, Zn3 mutations from Fig. 5 mapped onto the NMR structure.